Chased by wolves: Effects of a threat prime on working-memory in Portuguese and Hong Kong populations

Cultural differences between Western and East Asian populations have often been categorized in terms of Individualism versus Collectivism, or analytic versus holistic thinking and perception. In this study, 30 participants from Hong Kong and 26 participants from Portugal were compared. Individualism and Collectivism levels were measured using the Auckland Individualism and Collectivism Scale. Furthermore, they were tested on working memory using the Corsi Block-tapping task. Finally, they performed a modified version of the wolfpack task to investigate analytic / holistic perception, perceived animacy, and changes in working memory after a threat prime. We found an interaction between priming condition and culture. The performance of the Hong Kong group stayed constant when seeing a neutral prime and when seeing a threat prime. In contrast, the Portuguese group performed worse after seeing a threat prime, compared to a neutral prime. In both conditions, the Hong Kong group performed better than the Portuguese group. Hong Kong also scored higher on the Corsi block-tapping task, but this effect was only marginally significant.  While Hong Kong scored higher on Collectivism, no difference was found for Individualism. However, no correlation was found between Collectivism and working memory performance after threat prime. The wolfpack task used here proved insufficient to detect differences in analytic and holistic perception, or differences in perceived animacy across cultures. This study urges caution when applying the Individualism-Collectivism distinction to these cultures

Signal peptide peptidase-like 3 (SPPL3) is a type II membrane protein-selective sheddase that regulates cellular N-glycosylation

Intramembrane proteolysis - hydrolysis of membrane proteins within or close to their membrane-spanning regions - is a crucial cellular process that is conserved throughout all kingdoms of life. It is executed by distinct classes of polytopic membrane proteins, the intramembrane-cleaving proteases, that provide a hydrophilic, proteinaceous environment accommodating membrane protein substrates as well as water molecules within the hydrophobic membrane interior and catalyse peptide bond hydrolysis. In particular, intramembrane-cleaving aspartyl proteases have received attention as the presenilins, the catalytic subunits of the γ-secretase complex, were identified as key players in Alzheimer's disease pathophysiology. In addition to presenilins, mammalian genomes harbour presenilin homologues which include signal peptide peptidase (SPP) and SPP-like (SPPL) proteases. Among these, the Golgi-resident protease SPPL3 stands out as it is highly conserved among metazoa and SPPL3 orthologues are also found in plants. However, due to the lack of known substrates, SPPL3 has thus far hardly been characterised. Hence, the purpose of this study was to identify its substrates and elucidate its physiological function(s). In the first part of this study, the foamy virus envelope glycoprotein (FVenv) was identified as the first substrate of SPPL3. This allowed to study SPPL3's proteolytic activity in detail, with a focus on its substrate selectivity and sensitivity towards previously characterised inhibitors of intramembrane-cleaving aspartyl proteases. Importantly, this study revealed in addition that two other intramembrane-cleaving proteases, SPPL2a and SPPL2b, also endoproteolyse FVenv. SPPL2b in particular had been studied in detail before and therefore SPPL3- and SPPL2b- mediated endoproteolysis of FVenv were examined in parallel to directly compare these phylogenetically related intramembrane-cleaving proteases. This uncovered an unexpected idiosyncrasy of SPPL3 that clearly sets SPPL3 apart from other intramembrane-cleaving aspartyl proteases: SPPL3 endoproteolysed full-length FVenv and did not require the substrate's prior tailoring by another proteolytic activity - an otherwise common phenomenon among intramembrane-cleaving aspartyl proteases. In the second part, the physiological function of SPPL3 was investigated. Alterations in the cellular levels of proteolytically active SPPL3 turned out to impact the composition of N-glycans attached to endogenous cellular glycoproteins. SPPL3 over-expression was accompanied by a decrease in glycoprotein molecular weight, i.e. a hypoglycosylation phenotype, while loss of SPPL3 expression in cell culture models but also in vivo resulted in a hyperglycosylation phenotype. This led to the identification of Golgi glycan-modifying enzymes such as GnT-V and β3GnT1 as novel physiological substrates of SPPL3. Loss or reduction of SPPL3 expression, for instance, led to a marked intracellular accumulation of these enzymes, explaining the more extensive N-glycan elaboration and the hyperglycosylation phenotype observed under these conditions. At the same time secretion of these enzymes was reduced under these conditions. Together with additional observations such as the mapping of the SPPL3 cleavage site to the membrane-spanning region of GnT-V, this study demonstrates that SPPL3-mediated intramembrane proteolysis of such glycan-modifying enzymes liberates their active site-harbouring ectodomains. Acting in this manner, SPPL3 controls the intracellular pool of active glycan-modifying enzymes. Importantly, the finding that SPPL3 proteolytically cleaves full-length glycan-modifying enzymes and sheds their ectodomains is well in line with the observations made for FVenv and suggested that SPPL3 acts functionally equivalent to classical sheddases or rhomboid proteases but much unlike all other characterised mammalian intramembrane-cleaving aspartyl proteases. To examine whether these observations hold also true on a global cellular scale, a proteomic approach was undertaken in the third part of the study to define the SPPL3 degradome of HEK293 cells in conditions of SPPL3 over-expression. On the one hand, this led to the identification of numerous novel, mostly Golgi-resident candidate SPPL3 substrates and, considering the physiological implications, suggests that SPPL3 is very intricately linked to Golgi function. On the other hand, this approach supports the initial hypothesis that SPPL3 acts as a cellular type II membrane protein-selective sheddase. Taken together, this study provides the first in-depth characterisation of the intramembrane protease SPPL3 and reveals the cellular function of SPPL3. SPPL3 displays considerable and marked differences to other intramembrane-cleaving aspartyl proteases and emerges as a fundamental cellular sheddase that exhibits strong selectivity for type II-oriented, Golgi-resident membrane proteins. Products of SPPL3-mediated endoproteolysis of these Golgi factors are secreted and/or may be subject to intracellular degradation which compromises their catalytic activity. Thus, SPPL3 indirectly controls protein glycosylation in the Golgi apparatus.Intramembranproteolyse, die hydrolytische Spaltung von Membranproteinen innerhalb ihrer Transmembrandomänen, ist ein wichtiger zellulärer Prozess, der in allen Reichen der Lebewesen konserviert ist. Die Intramembranproteolyse wird von unterschiedlichen Klassen von polytopen Membranproteinen, den Intramembranproteasen, ausgeführt, die eine hydrophile Proteinumgebung innerhalb des hydrophoben Bereichs der Membran bilden. In dieser Umgebung finden sowohl Membranproteinsubstrate als auch Wassermoleküle Platz und Hydrolyse der Peptidbindung wird durch die Intramembranproteasen katalysiert. Vor allem die Aspartylintramembranproteasen haben sehr viel Aufmerksamkeit auf sich gezogen, da die Preseniline, die katalytischen Untereinheiten des γ-Sekretasekomplexes, als Schlüsselenzyme in der Pathophysiologie der Alzheimer-Erkrankung identifiziert wurden. Neben den Presenilinen finden sich in Säugetiergenomen auch Presenilinhomologe, welche sowohl die Signalpeptidpeptidase (SPP) als auch SPP-ähnliche (SPP-like, SPPL) Proteasen umfassen. Aus diesen sticht die im Golgi lokalisierte Protease SPPL3 heraus, da sie in vielzelligen Tieren hochkonserviert ist und sich SPPL3-Orthologe auch in Pflanzen finden. Gleichzeitig ist SPPL3 jedoch, vor allem aufgrund der Tatsache, dass keine Substrate beschrieben wurden, bisher kaum charakterisiert. Daher war es das Ziel dieser Arbeit Substrate von SPPL3 zu identifizieren und seine physiologische Funktion(-en) aufzuklären. Im ersten Teil dieser Arbeit wurde ein erstes SPPL3-Substrat, das foamy-virale Hüllglykoprotein (FVenv), identifiziert. Dies erlaubte die proteolytische Aktivität von SPPL3 und insbesondere seine Substratselektivität sowie seine Sensitivität gegenüber Inhibitoren von Aspartylintramem- branproteasen im Detail zu untersuchen. Es konnte ebenfalls festgestellt werden, dass zwei weitere Intramembranproteasen, SPPL2a und SPPL2b, FVenv ebenfalls endoproteolytisch spalten. Vor allem SPPL2b war bereits zuvor im Detail untersucht worden und daher konnte die SPPL3- beziehungsweise SPPL2b-vermittelte Endoproteolyse von FVenv verglichen werden. Dies offenbarte eine unerwartete Eigenheit von SPPL3, welche SPPL3 deutlich von anderen Intramembranproteasen, vor allem den anderen Aspartylintramembraneproteasen, unterschiedet: SPPL3 spaltete das FVenv-Holoprotein und bedarf keinem vorherigen Zuschneiden des Substrates durch eine andere proteolytische Aktivität - ein sonst sehr verbreitetes Phänomen bei Intramembranproteasen. Im zweiten Teil sollte die physiologischen Funktion von SPPL3 untersucht werden. Dabei zeigte sich, dass Änderungen in der zellulären SPPL3-Aktivität große Auswirkungen auf die Zusammensetzung der N-Glykane auf endogenen zellulären Glykoproteinen haben. SPPL3- Überexpression ging mit einer Reduktion des Molekulargewichts untersuchter Glykoproteine, einem so genannten Hypoglykosylierungsphänotyp, einher, während der Verlust der SPPL3- Expression im Zellkulturmodell aber auch in vivo in einem Hyperglykosylierungsphänotyp resultierte. Dies führte zur Identifizierung von glykanmodifizierenden Enzymen im Golgi wie beispielsweise GnT-V und β3GnT1 als physiologische SPPL3-Substrate. Verlust oder Reduktion der SPPL3-Expression führte zu einer starken intrazellulären Akkumulation dieser Substrate, was die unter diesen Bedingungen beobachtete umfassendere N-Glykan- ausarbeitung sowie den Hyperglykosylierungsphänotyp erklärte. Gleichzeitig wurde die Sekretion dieser Enzyme unter diesen Bedingungen stark beeinträchtigt. Zusammen mit weiteren Beobachtungen wie der Bestimmung der SPPL3-Schnittstelle im Bereich der Transmembrandomäne von GnT-V zeigte diese Studie, dass die von SPPL3 vermittelte Intramembranproteolyse solcher glykan-modifizierenden Enzyme deren Ektodomainen freisetzt und SPPL3 folglich auf diese Weise das intrazelluläre Reservoir aktiver glykan-modifizierender Enzyme kontrolliert. Hervorzuheben ist dabei, dass diese Erkenntnis in guter Übereinstimmung mit den für FVenv gemachten Beobachtungen steht. Dies legt wiederum nahe, dass SPPL3 in funktioneller Hinsicht mit einer klassischen Sheddase oder einer Rhomboidprotease gleichgestellt ist, sich jedoch von allen anderen charakterisierten Aspartylintramembranproteasen in Säugetieren unterscheidet. Um herauszufinden, ob diese Beobachtungen auch auf globaler zellulärer Ebene Bestätigung finden, wurde eine Proteomanalyse im dritten Teil dieser Arbeit durchgeführt, um das SPPL3-Degradom in HEK293-Zellen unter Überexpressionsbedingungen zu definieren. Dies führte einerseits zur Identifizierung vieler weiterer neuer, zumeist im Golgi lokalisierter SPPL3-Substratkandidaten und legte andererseits unter physiologischen Gesichtspunkten nahe, dass SPPL3 sehr mit der Funktion des Golgi-Apparats verwoben ist. Andererseits unterstützten diese Ergebnisse sehr deutlich die formulierte Hypothese, dass SPPL3 als zelluläre Typ-II-Membranprotein-selektive Sheddase agiert. Zusammengefasst liefert diese Arbeit die erste ausführliche Charakterisierung der Intramem- branprotease SPPL3. SPPL3 zeigt erhebliche Unterschiede zu anderen Aspartylintramembran- proteasen und erweist sich damit als bedeutende zelluläre Sheddase mit einer auffälligen Selektivität für Typ-II-orientierte, im Golgi lokalisierte Membranproteine. Produkte der Endoproteolyse dieser Golgi-Proteine werden sekretiert und/oder werden möglicherweise intrazellulär degradiert, was wiederum ihre eigentliche intrazelluläre Funktion beeinträchtigt. Folglich kontrolliert SPPL3 indirekt die Proteinglykosylierung im Golgi-Apparat

Identification of SH3 domain interaction partners of human FasL (CD178) by phage display screening

<p>Abstract</p> <p>Background -</p> <p>Fas ligand is a cytotoxic effector molecule of T and NK cells which is characterized by an intracellular N-terminal polyproline region that serves as a docking site for SH3 and WW domain proteins. Several previously described Fas ligand-interacting SH3 domain proteins turned out to be crucial for the regulation of storage, expression and function of the death factor. Recent observations, however, indicate that Fas ligand is also subject to posttranslational modifications including shedding and intramembrane proteolysis. This results in the generation of short intracellular fragments that might either be degraded or translocate to the nucleus to influence transcription. So far, protein-protein interactions that specifically regulate the fate of the intracellular fragments have not been identified.</p> <p>Results -</p> <p>In order to further define the SH3 domain interactome of the intracellular region of Fas ligand, we now screened a human SH3 domain phage display library. In addition to known SH3 domains mediating binding to the Fas ligand proline-rich domain, we were able to identify a number of additional SH3 domains that might also associate with FasL. Potential functional implications of the new binding proteins for the death factor's biology are discussed. For Tec kinases and sorting nexins, the observed interactions were verified in cellular systems by pulldown experiments.</p> <p>Conclusion -</p> <p>We provide an extended list of putative Fas ligand interaction partners, confirming previously identified interactions, but also introducing several novel SH3 domain proteins that might be important regulators of Fas ligand function.</p

Signal peptide peptidase-like 3 (SPPL3) is a type II membrane protein-selective sheddase that regulates cellular N-glycosylation

Intramembrane proteolysis - hydrolysis of membrane proteins within or close to their membrane-spanning regions - is a crucial cellular process that is conserved throughout all kingdoms of life. It is executed by distinct classes of polytopic membrane proteins, the intramembrane-cleaving proteases, that provide a hydrophilic, proteinaceous environment accommodating membrane protein substrates as well as water molecules within the hydrophobic membrane interior and catalyse peptide bond hydrolysis. In particular, intramembrane-cleaving aspartyl proteases have received attention as the presenilins, the catalytic subunits of the γ-secretase complex, were identified as key players in Alzheimer's disease pathophysiology. In addition to presenilins, mammalian genomes harbour presenilin homologues which include signal peptide peptidase (SPP) and SPP-like (SPPL) proteases. Among these, the Golgi-resident protease SPPL3 stands out as it is highly conserved among metazoa and SPPL3 orthologues are also found in plants. However, due to the lack of known substrates, SPPL3 has thus far hardly been characterised. Hence, the purpose of this study was to identify its substrates and elucidate its physiological function(s). In the first part of this study, the foamy virus envelope glycoprotein (FVenv) was identified as the first substrate of SPPL3. This allowed to study SPPL3's proteolytic activity in detail, with a focus on its substrate selectivity and sensitivity towards previously characterised inhibitors of intramembrane-cleaving aspartyl proteases. Importantly, this study revealed in addition that two other intramembrane-cleaving proteases, SPPL2a and SPPL2b, also endoproteolyse FVenv. SPPL2b in particular had been studied in detail before and therefore SPPL3- and SPPL2b- mediated endoproteolysis of FVenv were examined in parallel to directly compare these phylogenetically related intramembrane-cleaving proteases. This uncovered an unexpected idiosyncrasy of SPPL3 that clearly sets SPPL3 apart from other intramembrane-cleaving aspartyl proteases: SPPL3 endoproteolysed full-length FVenv and did not require the substrate's prior tailoring by another proteolytic activity - an otherwise common phenomenon among intramembrane-cleaving aspartyl proteases. In the second part, the physiological function of SPPL3 was investigated. Alterations in the cellular levels of proteolytically active SPPL3 turned out to impact the composition of N-glycans attached to endogenous cellular glycoproteins. SPPL3 over-expression was accompanied by a decrease in glycoprotein molecular weight, i.e. a hypoglycosylation phenotype, while loss of SPPL3 expression in cell culture models but also in vivo resulted in a hyperglycosylation phenotype. This led to the identification of Golgi glycan-modifying enzymes such as GnT-V and β3GnT1 as novel physiological substrates of SPPL3. Loss or reduction of SPPL3 expression, for instance, led to a marked intracellular accumulation of these enzymes, explaining the more extensive N-glycan elaboration and the hyperglycosylation phenotype observed under these conditions. At the same time secretion of these enzymes was reduced under these conditions. Together with additional observations such as the mapping of the SPPL3 cleavage site to the membrane-spanning region of GnT-V, this study demonstrates that SPPL3-mediated intramembrane proteolysis of such glycan-modifying enzymes liberates their active site-harbouring ectodomains. Acting in this manner, SPPL3 controls the intracellular pool of active glycan-modifying enzymes. Importantly, the finding that SPPL3 proteolytically cleaves full-length glycan-modifying enzymes and sheds their ectodomains is well in line with the observations made for FVenv and suggested that SPPL3 acts functionally equivalent to classical sheddases or rhomboid proteases but much unlike all other characterised mammalian intramembrane-cleaving aspartyl proteases. To examine whether these observations hold also true on a global cellular scale, a proteomic approach was undertaken in the third part of the study to define the SPPL3 degradome of HEK293 cells in conditions of SPPL3 over-expression. On the one hand, this led to the identification of numerous novel, mostly Golgi-resident candidate SPPL3 substrates and, considering the physiological implications, suggests that SPPL3 is very intricately linked to Golgi function. On the other hand, this approach supports the initial hypothesis that SPPL3 acts as a cellular type II membrane protein-selective sheddase. Taken together, this study provides the first in-depth characterisation of the intramembrane protease SPPL3 and reveals the cellular function of SPPL3. SPPL3 displays considerable and marked differences to other intramembrane-cleaving aspartyl proteases and emerges as a fundamental cellular sheddase that exhibits strong selectivity for type II-oriented, Golgi-resident membrane proteins. Products of SPPL3-mediated endoproteolysis of these Golgi factors are secreted and/or may be subject to intracellular degradation which compromises their catalytic activity. Thus, SPPL3 indirectly controls protein glycosylation in the Golgi apparatus.Intramembranproteolyse, die hydrolytische Spaltung von Membranproteinen innerhalb ihrer Transmembrandomänen, ist ein wichtiger zellulärer Prozess, der in allen Reichen der Lebewesen konserviert ist. Die Intramembranproteolyse wird von unterschiedlichen Klassen von polytopen Membranproteinen, den Intramembranproteasen, ausgeführt, die eine hydrophile Proteinumgebung innerhalb des hydrophoben Bereichs der Membran bilden. In dieser Umgebung finden sowohl Membranproteinsubstrate als auch Wassermoleküle Platz und Hydrolyse der Peptidbindung wird durch die Intramembranproteasen katalysiert. Vor allem die Aspartylintramembranproteasen haben sehr viel Aufmerksamkeit auf sich gezogen, da die Preseniline, die katalytischen Untereinheiten des γ-Sekretasekomplexes, als Schlüsselenzyme in der Pathophysiologie der Alzheimer-Erkrankung identifiziert wurden. Neben den Presenilinen finden sich in Säugetiergenomen auch Presenilinhomologe, welche sowohl die Signalpeptidpeptidase (SPP) als auch SPP-ähnliche (SPP-like, SPPL) Proteasen umfassen. Aus diesen sticht die im Golgi lokalisierte Protease SPPL3 heraus, da sie in vielzelligen Tieren hochkonserviert ist und sich SPPL3-Orthologe auch in Pflanzen finden. Gleichzeitig ist SPPL3 jedoch, vor allem aufgrund der Tatsache, dass keine Substrate beschrieben wurden, bisher kaum charakterisiert. Daher war es das Ziel dieser Arbeit Substrate von SPPL3 zu identifizieren und seine physiologische Funktion(-en) aufzuklären. Im ersten Teil dieser Arbeit wurde ein erstes SPPL3-Substrat, das foamy-virale Hüllglykoprotein (FVenv), identifiziert. Dies erlaubte die proteolytische Aktivität von SPPL3 und insbesondere seine Substratselektivität sowie seine Sensitivität gegenüber Inhibitoren von Aspartylintramem- branproteasen im Detail zu untersuchen. Es konnte ebenfalls festgestellt werden, dass zwei weitere Intramembranproteasen, SPPL2a und SPPL2b, FVenv ebenfalls endoproteolytisch spalten. Vor allem SPPL2b war bereits zuvor im Detail untersucht worden und daher konnte die SPPL3- beziehungsweise SPPL2b-vermittelte Endoproteolyse von FVenv verglichen werden. Dies offenbarte eine unerwartete Eigenheit von SPPL3, welche SPPL3 deutlich von anderen Intramembranproteasen, vor allem den anderen Aspartylintramembraneproteasen, unterschiedet: SPPL3 spaltete das FVenv-Holoprotein und bedarf keinem vorherigen Zuschneiden des Substrates durch eine andere proteolytische Aktivität - ein sonst sehr verbreitetes Phänomen bei Intramembranproteasen. Im zweiten Teil sollte die physiologischen Funktion von SPPL3 untersucht werden. Dabei zeigte sich, dass Änderungen in der zellulären SPPL3-Aktivität große Auswirkungen auf die Zusammensetzung der N-Glykane auf endogenen zellulären Glykoproteinen haben. SPPL3- Überexpression ging mit einer Reduktion des Molekulargewichts untersuchter Glykoproteine, einem so genannten Hypoglykosylierungsphänotyp, einher, während der Verlust der SPPL3- Expression im Zellkulturmodell aber auch in vivo in einem Hyperglykosylierungsphänotyp resultierte. Dies führte zur Identifizierung von glykanmodifizierenden Enzymen im Golgi wie beispielsweise GnT-V und β3GnT1 als physiologische SPPL3-Substrate. Verlust oder Reduktion der SPPL3-Expression führte zu einer starken intrazellulären Akkumulation dieser Substrate, was die unter diesen Bedingungen beobachtete umfassendere N-Glykan- ausarbeitung sowie den Hyperglykosylierungsphänotyp erklärte. Gleichzeitig wurde die Sekretion dieser Enzyme unter diesen Bedingungen stark beeinträchtigt. Zusammen mit weiteren Beobachtungen wie der Bestimmung der SPPL3-Schnittstelle im Bereich der Transmembrandomäne von GnT-V zeigte diese Studie, dass die von SPPL3 vermittelte Intramembranproteolyse solcher glykan-modifizierenden Enzyme deren Ektodomainen freisetzt und SPPL3 folglich auf diese Weise das intrazelluläre Reservoir aktiver glykan-modifizierender Enzyme kontrolliert. Hervorzuheben ist dabei, dass diese Erkenntnis in guter Übereinstimmung mit den für FVenv gemachten Beobachtungen steht. Dies legt wiederum nahe, dass SPPL3 in funktioneller Hinsicht mit einer klassischen Sheddase oder einer Rhomboidprotease gleichgestellt ist, sich jedoch von allen anderen charakterisierten Aspartylintramembranproteasen in Säugetieren unterscheidet. Um herauszufinden, ob diese Beobachtungen auch auf globaler zellulärer Ebene Bestätigung finden, wurde eine Proteomanalyse im dritten Teil dieser Arbeit durchgeführt, um das SPPL3-Degradom in HEK293-Zellen unter Überexpressionsbedingungen zu definieren. Dies führte einerseits zur Identifizierung vieler weiterer neuer, zumeist im Golgi lokalisierter SPPL3-Substratkandidaten und legte andererseits unter physiologischen Gesichtspunkten nahe, dass SPPL3 sehr mit der Funktion des Golgi-Apparats verwoben ist. Andererseits unterstützten diese Ergebnisse sehr deutlich die formulierte Hypothese, dass SPPL3 als zelluläre Typ-II-Membranprotein-selektive Sheddase agiert. Zusammengefasst liefert diese Arbeit die erste ausführliche Charakterisierung der Intramem- branprotease SPPL3. SPPL3 zeigt erhebliche Unterschiede zu anderen Aspartylintramembran- proteasen und erweist sich damit als bedeutende zelluläre Sheddase mit einer auffälligen Selektivität für Typ-II-orientierte, im Golgi lokalisierte Membranproteine. Produkte der Endoproteolyse dieser Golgi-Proteine werden sekretiert und/oder werden möglicherweise intrazellulär degradiert, was wiederum ihre eigentliche intrazelluläre Funktion beeinträchtigt. Folglich kontrolliert SPPL3 indirekt die Proteinglykosylierung im Golgi-Apparat

Posttranslational regulation of Fas ligand function

The TNF superfamily member Fas ligand acts as a prototypic death factor. Due to its ability to induce apoptosis in Fas (APO-1, CD95) expressing cells, Fas ligand participates in essential effector functions of the immune system. It is involved in natural killer cell- and T cell-mediated cytotoxicity, the establishment of immune privilege, and in termination of immune responses by induction of activation-induced cell death. In addition, Fas ligand-positive tumours may evade immune surveillance by killing Fas-positive tumour-infiltrating cells. Given these strong cytotoxic capabilities of Fas ligand, it is obvious that its function has to be strictly regulated to avoid uncontrolled damage. In hematopoietic cells, the death factor is stored in secretory lysosomes and is mobilised to the immunological synapse only upon activation. The selective sorting to and the release from this specific lysosomal compartment requires interactions of the Fas ligand cytosolic moiety, which mediates binding to various adapter proteins involved in trafficking and cytoskeletal reorganisation. In addition, Fas ligand surface expression is further regulated by posttranslational ectodomain shedding and subsequent regulated intramembrane proteolysis, releasing a soluble ectodomain cytokine into the extracellular space and an N-terminal fragment with a potential role in intracellular signalling processes. Moreover, other posttranslational modifications of the cytosolic domain, including phosphorylation and ubiquitylation, have been described to affect various aspects of Fas ligand biology. Since FasL is regarded as a potential target for immunotherapy, the further characterisation of its biological regulation and function will be of great importance for the development and evaluation of future therapeutic strategies

Probing Plasmodium falciparum sexual commitment at the single-cell level

Background: Malaria parasites go through major transitions during their complex life cycle, yet the underlying differentiation pathways remain obscure. Here we apply single cell transcriptomics to unravel the program inducing sexual differentiation in Plasmodium falciparum. Parasites have to make this essential life-cycle decision in preparation for human-to-mosquito transmission. Methods: By combining transcriptional profiling with quantitative imaging and genetics, we defined a transcriptional signature in sexually committed cells. Results: We found this transcriptional signature to be distinct from general changes in parasite metabolism that can be observed in response to commitment-inducing conditions. Conclusions: This proof-of-concept study provides a template to capture transcriptional diversity in parasite populations containing complex mixtures of different life-cycle stages and developmental programs, with important implications for our understanding of parasite biology and the ongoing malaria elimination campaign

A combinational approach of multilocus sequence typing and other molecular typing methods in unravelling the epidemiology of Erysipelothrix rhusiopathiae strains from poultry and mammals

Erysipelothrix rhusiopathiae infections re-emerged as a matter of great concern particularly in the poultry industry. In contrast to porcine isolates, molecular epidemiological traits of avian E. rhusiopathiae isolates are less well known. Thus, we aimed to (i) develop a multilocus sequence typing (MLST) scheme for E. rhusiopathiae, (ii) study the congruence of strain grouping based on pulsed-field gel electrophoresis (PFGE) and MLST, (iii) determine the diversity of the dominant immunogenic protein SpaA, and (iv) examine the distribution of genes putatively linked with virulence among field isolates from poultry (120), swine (24) and other hosts (21), including humans (3). Using seven housekeeping genes for MLST analysis we determined 72 sequence types (STs) among 165 isolates. This indicated an overall high diversity, though 34.5% of all isolates belonged to a single predominant ST-complex, STC9, which grouped strains from birds and mammals, including humans, together. PFGE revealed 58 different clusters and congruence with the sequence-based MLST-method was not common. Based on polymorphisms in the N-terminal hyper-variable region of SpaA the isolates were classified into five groups, which followed the phylogenetic background of the strains. More than 90% of the isolates harboured all 16 putative virulence genes tested and only intI, encoding an internalin-like protein, showed infrequent distribution. MLST data determined E. rhusiopathiae as weakly clonal species with limited host specificity. A common evolutionary origin of isolates as well as shared SpaA variants and virulence genotypes obtained from avian and mammalian hosts indicates common reservoirs, pathogenic pathways and immunogenic properties of the pathogen

Electrocardiologic and related methods of non-invasive detection and risk stratification in myocardial ischemia: state of the art and perspectives

Background: Electrocardiographic methods still provide the bulk of cardiovascular diagnostics. Cardiac ischemia is associated with typical alterations in cardiac biosignals that have to be measured, analyzed by mathematical algorithms and allegorized for further clinical diagnostics. The fast growing fields of biomedical engineering and applied sciences are intensely focused on generating new approaches to cardiac biosignal analysis for diagnosis and risk stratification in myocardial ischemia